J. Microbiol. Biotechnol. (2013), 23(4), 511–517http://dx.doi.org/10.4014/jmb.1209.09066First published online February 2, 2013pISSN 1017-7825 eISSN 1738-8872

Ethanol Production by Repeated Batch and Continuous Fermentations bySaccharomyces cerevisiae Immobilized in a Fibrous Bed Bioreactor

Chen, Yong1,2†

, Qingguo Liu1,3†

, Tao Zhou1,3

, Bingbing Li1,3

, Shiwei Yao1,3

, An Li1,3

, Jinglan Wu1,3

, and

Hanjie Ying1,2,3

*†

1College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, China2National Engineering Technique Research Center for Biotechnology, Nanjing, China3State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing, China

Received: September 25, 2012 / Revised: December 21, 2012 / Accepted: December 26, 2012

In this work, a fibrous bed bioreactor with high specific

surface area and good adsorption efficacy for S. cerevisiae

cells was used as the immobilization matrix in the

production of ethanol. In batch fermentation, an optimal

ethanol concentration of 91.36 g/l and productivity of

4.57 g l-1

h-1

were obtained at an initial sugar concentration

of 200 g/l. The ethanol productivity achieved by the

immobilized cells was 41.93% higher than that obtained

from free cells. Ethanol production in a 22-cycle repeated

batch fermentation demonstrated the enhanced stability

of the immobilized yeast cells. Under continuous fermentation

in packed-bed reactors, a maximum ethanol concentration

of 108.14 g/l and a productivity of 14.71 g l-1

h-1 were

attained at 35oC, and a dilution rate of 0.136 h

-1 with 250 g/l

glucose.

Key words: Immobilization, ethanol fermentation, fibrous

bed bioreactor

Ethanol is a specialty chemical with many applications in

the chemical industry as well as in foodstuffs and medical

treatments. It can also be used as a renewable and

alternative form of bioenergy and is currently under

intensive study for this purpose [11]. Traditional ethanol

fermentation industries produce ethanol by batch or fed-

batch processes. Compared with suspended-cell fermentation,

the immobilization of microbial cells has many attractive

advantages, such as increasing the biomass in the reactor

and the productivity of fermentation, preserving the

activity of cells, and affording the possibility of continuous

fermentation, among others [10, 20]. However, conventional

immobilized-cell fermentation usually suffers from

productivity loss due to toxicity or poor stability of carriers

such as calcium polyurethane foam, alginate, gelatin, and

κ-carrageenan [1, 14].

Recently, a fibrous bed bioreactor (FBB) with cells

immobilized in a fibrous matrix packed in the reactor has

been successfully used to produce several organic products,

such as lactic acid [28], butyric acid [34], butanol [12], and

mycophenolic acid [32], due to its high specific surface

area, high porosity, low cost, strong stability, and good

adsorption efficacy. In addition, a FBB can also offer

protection of the cells against mechanical shear stress [27]

and continuously renew the cell population [8]. Kilonzo

et al. [15, 16] observed a production of glucoamylase

obtained by immobilizing recombinant Saccharomyces

cerevisiae in a FBB using a cotton fabric. However, only a

few articles have referred to the use of immobilized yeast

in a FBB for ethanol production.

This study on ethanol fermentation has been focused

primarily on comparing the fermentation performances of

immobilized and free cells in batch culture and ascertaining

the stability of the immobilized cells in repeated batch

fermentation. Continuous fermentation in packed-bed

reactors was also investigated.

MATERIALS AND METHODS

Microorganism and Culture Medium

Industrial Saccharomyces cerevisiae 1308 was supplied by Tian-

Guan Group (Henan, China). It was stored on a malt juice agar slant

that contained 250 g/l malt and 20 g/l agar at 4oC and cultured on

malt juice agar slant for 2 days at 30oC before use.

Starter cultures were prepared by transferring a loopful of the

stock culture to 200 ml of the sterilized preculture medium, which

*Corresponding authorPhone: +86-25-86990001; Fax: +86-25-58139389;E-mail: yinghanjie@njut.edu.cn†These authors contributed equally to this work.

512 Chen et al.

was composed of 10 g/l yeast extract, 20 g/l peptone, 20 g/l glucose,

1.5 g/l KH2PO4, 4 g/l (NH4)2SO4, and 0.5 g/l MgSO4. The cell

cultivation was carried out on a shaker at 150 rpm and 30oC for

about 18 h.

The fermentation medium used for microorganism culture contained

(per L) 200 g glucose, 4 g peptone, 3 g yeast extract, 4 g (NH4)2SO4,

3 g KH2PO4, 0.05 g FeSO4·7H2O, 0.05 g ZnSO4·7H2O, and 0.5 g

MgSO4. The initial pH was 5.17 without adjustment, and the

medium was sterilized at 115oC for 15 min.

Cell Immobilization

Cells were immobilized as follows: 15 g of fibrous matrix (described

in the section below) and 200 ml of culture medium were added to a

500 ml Erlenmeyer flask and autoclaved for 15 min at 115oC. Then,

20 ml of stock cell suspension was added to the sterilized culture

medium and incubated at 100 rpm and 30oC to induce natural cell

adhesion for 48 h, when the OD value declined to 1 in the solution.

The fermented liquid was decanted and the support was wrung out

gently and then used for ethanol production.

Batch Fermentation

Batch fermentations were performed in 500 ml flasks containing

200 ml of fermentation medium and the immobilized cells or free

cells (free cells were incubated as long as immobilized cells and

were then collected by centrifuging at 5,000 ×g for 10 min and

washed twice with sterile water). The flasks were then inoculated in

a rotary shaker at 150 rpm and 35oC. All experiments were carried

out in triplicate.

Repeated Fermentation

The repeated batch fermentations were first carried out in batch

mode. At the end of each batch (namely, no glucose was determined),

the fermented broth was drained and only the carrier with

immobilized cells was retained in the flask. Then, the same amount

of the fermentation medium was immediately replaced to start the

next cycle. Twenty-two cycles were performed, and all experiments

were also carried out in triplicate.

Continuous Fermentation

Fibrous bed bioreactor. The fibrous materials used in this study

were the woven cotton fabric. The cotton matrix (thickness of 0.30

cm, specific surface area of 42 m2/m3, and porosity of 96%) was

bought from Weifang Hao Embellish Textile Co., Ltd, China. A

stainless steel wire cloth (SUS 304; porosity 0.97, mesh no. 50;

Hisin Wire Mesh Products Co., Ltd, China) topped with the fibrous

cotton sheets (60.00 cm × 30.50 cm × 0.30 cm and 120 g) was wound

into a spiral configuration along the vertical axis, just as described

by Kilonzo [16].

Fig. 1 shows the experimental setup of the continuous FBB system.

The FBB system was made of five stainless steel fermentation tanks

(each of working volume 1.6 L) that were linked in series and

packed with spiral wound cotton matrix. Before use, the reactor was

sterilized at 121oC for 1 h and then cooled to room temperature. The

stock cell suspension was then introduced at the bottom of the first

reactor and returned from the top outlet of the last reactor to the

culture vessel for re-aeration at a flow rate of 10 ml/min. After about

24 h of continuous circulation, most of the cells were immobilized,

and the cell density declined quite slowly. Then, the flow was

stopped and the medium was replaced by the fermentation medium,

which was continuously fed at a dilution of 0.5 h-1 to allow the

medium to fill all of the reactors quickly until they were full.

Fermentation kinetics study. The fermentation kinetics, mainly in

the solution phase, was determined with the FBB fed continuously

with fermentation medium. The effect of the dilution rate on the

fermentation was first examined at 25oC and 200 g/l glucose by

varying the dilution rate. The effect of temperature was then

investigated for three different temperatures at a dilution rate of

0.136 h-1 and 200 g/l glucose. Finally, measurements were made for

a high initial concentration of glucose under conditions of 35oC and

a dilution rate of 0.136 h-1. In all cases, the conditions were not

changed until the fermentation had reached a steady state.

Analytical Methods

At specified time intervals, 2 ml of sample was collected. The free

cells were determined by measuring the optical density (OD) at 660

nm using a UV-visible spectrophotometer (CA, USA) and by

applying the gravimetric method (dry cell weight). The immobilized

cells in the FBB were removed from the fibrous matrix by vortexing

the matrix and then estimated by measuring the OD value. The

values of the absorbance were converted to the cell dry weight

(DCW) using a calibration curve: 1 OD = 0.81 g DCW/l.

After removing the cells by centrifuging at 12,000 ×g for 3 min,

the clear fermentation broth was used to analyze for residual glucose,

ethanol, and glycerol by high-performance liquid chromatography

(HPLC) with a BIO-Rad HPX-87H organic acid analysis column at

25oC, using 0.05 N H2SO4 as the eluent at a flow rate of 0.6 ml/min.

RESULTS AND DISCUSSION

Batch and Repeated Batch Fermentation

Comparisons of free and immobilized cells in the batch

fermentation. Preliminary studies have shown typical

kinetics for batch fermentations with free cells and

immobilized cells in the fibrous bed bioreactor. As seen in

Fig. 2, the OD value of and ethanol produced by the free

cell system increased as glucose decreased, and the former

remained constant after 20 h. The OD value of and ethanol

produced by the immobilized cell system also increased as

glucose decreased at the initial stage of fermentation. The

Fig. 1. Schematic diagram of the immobilized-cell packed-bedreactors: 1, feed tank; 2, pump; 3, bioreactor; 4, water bath; 5,product tank.

ETHANOL PRODUCTION BY SACCHAROMYCES CEREVISIAE 513

OD reached a maximum value at 10 h, but then decreased

because of decreasing nutrition and adsorption by the

FBB. However, the final total biomass concentration of the

FBB system was much higher than that of the free system

(Table 1). This result indicated that cell immobilization could

increase the total cell concentration. In the FBB system,

yeast cells attached to the support and may form thick

layers of cells known as “biofilm.” The biofilm cells

produce extracellular polymeric substances (EPS) that

provide protection by providing a diffusive barrier to any

toxic compounds that could harm the cells, which results in

cell densities in the support increasing [9, 23]. Meanwhile,

from the table, we can see that the final ethanol concentration

and the yield (grams of ethanol produced per gram of

glucose consumed) obtained from FBB-immobilized cells

(FBB-cells) were similar to those from the free cells.

However, the ethanol productivity (concentration of ethanol

produced per hour) of the immobilized cells was 41.93%

higher than that of the free cells, which may be due to the

increased cell concentration.

Stability of immobilized yeast cells in repeated batch

fermentation. To investigate the stability of the FBB as a

support material for cell immobilization and the ability of

the immobilized cells to produce ethanol, a 22-cycle repeated

batch fermentation with S. cerevisiae 1308 immobilized on

FBB was carried out. As shown in Fig. 3, the residual

glucose was almost completely consumed by immobilized

cells within 8 h after the second batch. The ethanol production

and yield were found to be almost the same (91.57 ±

1.71 g/l, 0.457 ± 0.009 g/g) in the 22-cycle fermentation.

The productivity was improved from 4.57 to 11.31 g l-1 h-1

in the first 3 cycles of batch fermentation, due to the

increased biomass attached to the FBB. After the third

cycle of fermentation, because the FBB was saturated with

cells, the productivity reached a steady state, and the

average value was 11.75 g l-1 h-1, which was much higher

than the productivities reported by others (Table 2).

These results suggest that the FBB enhanced the

productivity and provided sufficient stability for immobilized-

cell fermentation. The increased ethanol productivity was

consistent with an increase of yeast cells adsorbed on the

inner/outer surfaces during the process, because ethanol is

a typical primary metabolite during which cell growth

also occurs. In addition, the time reduction during the

fermentation of successive batches process may be due to

the diffusion limitation having been overcome by surface

Fig. 2. Comparisons of the fermentation performances of freeand immobilized cells in batch culture. (A) Free-cell fermentation. (B) Immobilized-cell fermentation in the FBB.

Table 1. Comparison of the final parameters of the free and immobilized yeast cells in batch fermentation.

Residual sugar(g/l)

Ethanol(g/l)

Total biomass(g/l)

Productivity(g l-1 h-1)

Yield(g/g)

Free fermentation 0.80 90.14 30.80 3.22 0.453

Immobilized fermentation 0.93 91.36 41.52a 4.57 0.459

a

41.52 represents the value of the total cell concentration in the FBB system, which includes free and immobilized cells. The methods of detection and

calculation are described in the Materials and Methods section.

Fig. 3. Stabilization of the immobilized yeast cells by the FBB inrepeated batch fermentation.

514 Chen et al.

growth of cells [3]. In the repeated batch process, we found

no lag time for ethanol production at the start of each

batch, indicating that the immobilized cells were vigorous

throughout the experiment, just as reported by Watanabe et

al. [31]. The enhanced cell stability of the immobilized cell

culture may imply that an accumulation of saturated fatty

acids, which might be gradually increased with cell reuse,

leads to greater ethanol tolerance by increasing the fluidity

of the plasma membranes [33]. Moreover, the carrier may

have protected the yeast cells from toxic conditions, such

as acetic acid [17], during the fermentation of successive

batches.

Continuous fermentation

The effects of dilution rate. Ethanol production using

FBB cells was further examined by continuous fermentation

in a series-wound bioreactor. The reactors were operated

by gradually decreasing the dilution rate from 0.136 h-1

initially to 0.091 h-1 at 52 h and 0.045 h-1 at 88 h. As can be

seen in Fig. 4, the OD value and outlet concentrations of

substrate and products changed as the dilution rate

changed and, in general, they achieved a steady state at the

end of each period of constant operating conditions. In the

early stage, the OD reached the maximum value, due to

suspended cells that had leaked from the carrier by

oscillations during which the fresh medium been fed, and

then rapidly decreased because of adsorption and loss.

Besides this, we found the outlet concentrations of ethanol

and glycerol increased gradually with glucose decreased,

and after 40 h, all concentrations became almost constant.

When the dilution rate decreased from 0.136 to 0.091 h-1,

the OD value had no increase, and this may be because the

new growth of cells was compensated for adsorption by

the carriers. The outlet concentrations of glucose, ethanol,

and glycerol changed as before. When the dilution rate was

adjusted to 0.045 h-1, the OD value had a little improvement

because the growth rate was faster than the adsorption rate.

The highest concentrations of ethanol and glycerol produced

in the fermentation were 95.0 and 6.23 g/l, respectively, when

the glucose was exhausted after 128 h. In contrast, low

ethanol and glycerol concentrations of 35.9 and 4.33 g/l

were obtained at the higher dilution rate of 0.136 h-1. This

may be explained by the fact that, at a higher dilution rate,

the medium had a shorter contact time with the cells [12]

and, in addition, the biomass concentration was reduced by

wash-out due to the higher dilution rate [25] so that less

glucose was consumed and less products were produced in

the fermentation.

The reactor volumetric productivities (and yields as

analyzed below) at 25oC and various dilution rates were

estimated based on the time course data in the steady state

and are shown in Table 3. In general, the productivities for

ethanol and glycerol decreased as the dilution rate

decreased from 0.136 to 0.045 h-1, which is in agreement

with data published by others [12, 21, 20]. This phenomenon

could be explained by a decline in the effect of mass

transfer and the inhibition of cell activity by the higher

ethanol concentration [2, 25] as the dilution rate decreased.

At the same time, less oxygen was conveyed to the

bioreactors from the feed pump at the lower dilution rate,

which exacerbated the inhibition by ethanol [26]. Therefore,

the maximum ethanol (4.88 g l-1 h-1) and glycerol (0.59 g l-1 h-1)

productivities were obtained at a dilution rate of 0.136 h-1,

whereas lower productivities for ethanol and glycerol were

obtained at the lower dilution rate.

In terms of the effect of dilution rate on ethanol yield,

the lower dilution rate gave a higher yield than the high

dilution rate owing to a more complete reaction [12],

Table 2. Comparison of repeated batch ethanol fermentation with yeast cells immobilized on various carriers.

CarrierInitial sugar

(g/l)Residual sugar

(g/l)Batches

Ethanol(g/l)

Yield(g/g)

Productivity(g l

-1h-1)

Cashew apple bagasse [22] 83.29 3.30 10 37.83 0.454 6.28

Silk cocoons [24] 240.00 52.12 5 88.30 0.470 1.84

Sweet sorghum stalk [4] 230.00 8.17 8 104.26 0.470 1.43

Corncob [18] 230.00 27.52 8 97.19 0.480 2.02

Sorghum bagasse [33] 200.00 0 21 96.00 0.480 5.72

Calcium alginate [13] 150.00 4.00 7 73.50 0.50 6.10

Fibrous bed reactor (this work) 200.00 0 22 91.57 0.458 11.75

Fig. 4. Kinetics of continuous ethanol fermentation at 25oC,200 g/l glucose and various dilution rates.

ETHANOL PRODUCTION BY SACCHAROMYCES CEREVISIAE 515

which was in contrast to the trend for the glycerol yield. As

can be seen from Table 3, the maximum value of the

ethanol yield was 0.476 g/g at a dilution rate of 0.045 h-1,

and the minimum value was 0.460 g/g at a dilution rate of

0.136 h-1, whereas the maximum and minimum of glycerol

yields were 0.056 g/g at a dilution rate of 0.136 h-1 and

0.031 g/g at a dilution rate of 0.045 h-1, respectively.

The effects of temperature. After 136 h, the dilution rate

was increased from 0.045 to 0.136 h-1, and ethanol decreased

and glucose increased. When the temperature was adjusted

to 30oC at 184 h, the OD value and the concentration of

ethanol increased and glucose obviously decreased. After

the temperature was adjusted to 35oC, ethanol increased to

93.6 g/l, while glucose declined to about 4 g/l by 240 h

(Fig. 5). However, the concentration of glycerol was

evidently not affected throughout the process. From the

kinetics, we know that increasing the temperature not only

improves the enzyme activity, but also promotes new cell

growth.

Table 4 shows the productivities and product yields that

were estimated at the dilution rate of 0.136 h-1 and various

temperatures. In general, the productivity for ethanol increased

with increasing temperature and reached a maximum value

of 12.78 g l-1 h-1 at 35oC, while the productivity for glycerol

increased slowly and the maximum value was 0.93 g l-1 h-1.

Furthermore, when the temperature was adjusted to 30oC

from 25oC, it affected the productivities more than when

the temperature was adjusted from 30oC to 35oC, because

the high ethanol concentration produced at 35oC enhanced

the inhibitory effect. The effect of temperature on the

ethanol yield was also obvious. The maximum value was

0.480 g/g, acquired at 35oC, which was far higher than that

at 25oC. However, the glycerol yield showed the opposite

tendency in the range studied.

The effects of high initial concentration of glucose. High

ethanol concentrations have been continuously pursued in

the industry because they reduce the energy consumed

during distillation and waste distillage treatment [5, 6]. It

has been possible to make the final ethanol concentration

increase dramatically owing to the high ethanol tolerance

of S. cerevisiae [30]. Laopaiboon et al. [19] attained an

ethanol concentration of 120 g/l by fermenting sweet

Table 3. Effects of dilution rate on fermentation parameters at 25oC and 200 g/l glucose.

Dilution(h-1)

Residual sugar(g/l)

Ethanol(g/l)

Glycerol(g/l)

Productivity (g l-1 h-1) Yield (g/g)

Ethanol Glycerol Ethanol Glycerol

0.136 122.10 35.90 4.33 4.88 0.59 0.460 0.056

0.091 94.80 49.40 4.99 4.50 0.45 0.470 0.047

0.045 0.40 95.0 6.23 4.27 0.28 0.476 0.031

Fig. 5. Kinetics of continuous ethanol fermentation at a dilutionrate of 0.136 h

-1, 200 g/l glucose, and various temperatures.

Table 4. Effects of temperature on the fermentation parameters at a dilution rate of 0.136 h-1 and 200 g/l glucose.

Temperature(oC)

Residual sugar(g/l)

Ethanol(g/l)

Glycerol(g/l)

Productivity (g l-1 h-1) Yield (g/g)

Ethanol Glycerol Ethanol Glycerol

25 69.00 60.50 6.40 8.23 0.87 0.462 0.049

30 16.90 86.90 6.75 11.82 0.92 0.475 0.037

35 4.10 94.00 6.81 12.78 0.93 0.480 0.035

Fig. 6. Kinetics of continuous ethanol fermentation at 35oC, dilution

rate of 0.136 h-1, and various high initial sugar concentrations.

516 Chen et al.

sorghum juice. However, the productivity was very low,

which limited the feasibility of industrial production.

In this paper, in order to improve the high-gravity

fermentation by immobilized yeast cells, different high

initial sugar concentrations (200, 250, and 300 g/l) were

analyzed (Fig. 6). It can be seen that the ethanol

concentration increased as the initial sugar concentration

increased from 200 to 250 g/l, reaching a value of

108.14 g/l, but decreased at the initial sugar concentration

of 300 g/l. The initial sugar utilization was improved by

25% from 200 g/l, but the practical utilized sugar was

improved by 20%, which was less than the theoretical

value. In addition, when the initial sugar concentration was

adjusted to 300 g/l, the practical utilized sugar was

improved by 11%, which was much less than when the

initial concentration was 200 g/l. This might be due to the

osmotic pressure as described by Laopaiboon et al. [19],

which causes inhibition of cell activities and biomass. In

addition, the high concentration of glucose led to a high

ethanol concentration and increased the amount of CO2

generated, which may be another inhibiting factor. Beyond

the above, Bai et al. [6] found that the negative impact of

high temperature on the ethanol fermentation performance

was much worse under the VHG conditions than the

regular fermentation.

Table 5 indicates that a high initial sugar concentration

had significant effects on the main fermentation parameters.

When the initial sugar concentration increased from 200 to

250 g/l, the productivity correspondingly increased, and

when it was adjusted to 300 g/l, the productivity declined.

However, the glycerol concentration and productivity

always exhibited rising trends. The maximum values of

the ethanol and glycerol productivities were 14.71 g l-1 h-1

at 250 g/l glucose and 1.26 g L-1 h-1 at 300 g/l glucose,

respectively.

The yield of ethanol obviously decreased as the initial

sugar concentration increased. The maximum and minimum

values were 0.480 g/g at 200 g/l and 0.451 g/g at 300 g/l,

respectively. This may be explained by the fact that

glucose was used to form by-products such as glycerol,

acetaldehyde, and acetate, and some of these have inhibitory

effects on ethanol fermentation [6].

In conclusion, we have demonstrated the potential use of

S. cerevisiae 1308 immobilized by FBB for repeated batch

and continuous ethanol fermentations. Many advantages,

such as reusability, good adsorption efficacy, and high cell

density, were achieved and resulted in stable operation

with a high ethanol productivity and high yield [10]. A

comparison of the batch and repeated batch fermentations

showed that the FBB system was faster and more stable

than the conventional free fermentation system. A further

study of the continuous fermentation in a series of five

reactors showed that progressively decreasing dilution

rates can enhance the efficiency of ethanol production in

terms of ethanol concentration and product yield; however,

low productivity was obtained. Glycerol, which acts as a

by-product, was also affected greatly by the dilution rate.

The temperature and high initial sugar concentration

mainly affected the activities of the cells and biomass and,

finally, had an evident effect on the concentrations of

products, productivities, and yields.

Acknowledgments

This work was supported by a grant from the National

Outstanding Youth Foundation of China (Grant No.

21025625); the National High-Tech Research and Development

Program of China (863) (Grant No. 2012AA021203); the

National Basic Research Program of China (973) (Grant

No. 2011CBA00806); the National Natural Science

Foundation of China, Youth Program (Grant No. 21106070);

the Changjiang Scholars and Innovative in University

(PCSIRT); and the Project Funded by the Priority Academic

Program Development of Jiangsu Higher Education

Institutions (PAPD).

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